Solvent extraction of penicillin



Patented Sept. 27, 1949 SOLVENT EXTRACTION F PENICILLIN Derrick Rowley,London, Herbert Steiner, Eccles, and Emanuel Zimkin, Sale, England,assignors to Petrocarbon Limited, London, England No Drawing.Application May 16, 1946, Serial No. 670,316. In Great Britain September26,

This invention relates to the solvent extraction of penicillin fromaqueous solution and in particular to its extraction from fermentedbroth, though it is notlimited thereto but comprises extraction from anyother aqueous medium.

By the term penicillin, we denote the group of anti-bacterial agentswhich, as Fleming has shown, are produced by certain moulds probably byPenicillz'um notatum (see British Journal of Experimental Pathology,1929, vol. 10, p. 226).

In the usual method of producing penicillin the latter is recovered fromfermented solutions by extraction with solvents. This solvent extractionfulfils two purposes. It concentrates the penicillin into a smalleramount of solvent and thus reduces the volume which has to be evaporatedfinally for the recovery of the solid penicillin concentrate, andsecondly selective solvents extract penicillin in preference to itsaccompanying impurities and thus allow one to recover a much purerproduct.

There are, however, a number of features asso ciated with that processwhich render 'diflicult a quantitative recovery and the production of apenicillin of high purity.

The object of the present invention is to provide an improved method forthe solvent extrac tion of penicillin from aqueous media.

The extraction of penicillin is based on the fact that penicillin is aweak acid which, in aqueous solutions of increasing acidity (pl-I lessthan 4) becomes less and less dissociated. and thus more soluble inorganic solvents. From solutions of pH not less than 6, on the otherhand, it is easy to extract the penicillin from organic solvents intothe aqueous phase.

This procedure of extracting into solvents and l e-extracting fromsolvents is usually carried out twice, although to obtain a pure productthe procedure can be repeated more frequently. than one solvent may beused, that is to say, if for instance amylacetate is used in the firstextraction chloroform is frequently used in the second extraction, andfurther solvents may be used in further stages. In any case thereresults finally a dilute solution of the sodium or calcium salt ofpenicillin, depending on whether sodium hydroxide or calcium hydroxideis used in the final neutralising extraction. This solution stillcontains a considerable amount of impurities, thus the solid obtainedfrom it by evaporation in vacuo is by no means the pure salt 0fpenicillin. The evaporation is done in practice usually by the method offreeze drying and there results a product of a purity of about 300-900Oxford units per milli- 3 Claims. (Cl. 260302) 2 gram (Lancet, 1941,177) which assuming that pure penicillin on an average corresponds toabout 1600 O. ILL/mg. is equivalent to a purity of from 20 to 60 percent.

The yield of penicillin is about 50-80% of the penicillin initiallypresent in the broth.

While at the'higher limit these results seem reasonably satisfactory atthe lower limit recoveries and purity are poor. Yet even with solventswhich, according to their distribution characteristics', should giveresults near or even better than the best of those mentioned above, itis by no means possible to obtain them continuously. The reason for thisis the lability of the penicillin particularly in acidic solution. Toillustrate this point it has ben found that in a solution of penicillincontaminated with the usual impurities kept at 10 C. and at a pH of 2half the penicillin present initially is inactivated within a period ofabout 20 minutes while at a pH of 3 the penicillin is already much morestable, minutes being required to reduce its potency to one half of itsoriginal value. In order to carry out the extraction in an efilcientandeconomic way it is necessary to extract it from the aqueous solution ata pH of about 2, since at values of pH greater than 2 the extractioncoefficients of the usual solvents such as amylacetate ormethyl-isobutylketone are too unfavourable. In order to avoiddestruction of penicillin it has heretofore been necessary to employsuch types of apparatus as extract efficiently within a very short timeof contact. This apparatus, however, is expensive and difficult toconstruct. Even with such apparatus trivial incidents such'as partialstopping of tubes or passages may occur which involve unduly long con--tact of the penicillin and under pH conditions which are deleterious tothe penicillin. It is therefore not surprising that under thoseconditions, even with'the best equipment, quite frequently large lossesof penicillin occur due to inactivation. 7

We have foundthat these conditions of having to work at a dangerous pHcan be avoided by using certain types of solvents which have aconsiderably improved solvent power as compared with the best solventsused hitherto. This improved solvent power allows one to work at a pHgreater than 2 and thereby give a larger margin of safety and thus avoidthe difiiculties mentioned above. It is quite easy to work at a pH of 4and if several extraction stages are used it is possible to work at a pHof 5 and even approaching 6. The solvents have the further advantage ofallowing an increased reduction in volume of the solution containingpenicillin and they possess good selectivity, which allows one torecover a penicillin of considerable purity.

Apart from great solvent power an efficient solvent must be selectivefor penicillin, that is, it mustudissolve spenicillin inppreferencetothe impurities. accompanying it. iOf'theseiimpui'ities various organicacids are the most difiicult to remove. The solvents of the presentinvention exhibit selective properties which in, gen eral are betterthan thosepossessed', -for -instance, by amylacetate, althoughnotmecessarily as specific as chloroform. =';.hlcrofor=mehowever has thedrawback of a low solventirpoweroparticularly at a pH of 2.5consequently if it. is used, 1

one has to extract at low an withitheattending dangers of inactivation.

The invention consists in a process for solvent extraction of penicillinwherein the solvent or solvents, which may; be single or mixedsolvefits,:employed.- belong' to the! EIESSHCOHSYSfiHg-Dfthatmethylcyclohexaznones, ire. moncmethylaarrd ldimethylcyclohexanones.

(1) The process is applied totheextractiontef penicillin. from efermente'd broth;

(2) Theqorocessiziszappliedf'totthe"purification :of' apenicil-linby'repeatd'rextraction aandzrr'e extraction;

1 t3) :The process emayeemploy solvents which are -themselves 'm'ixturesoof isomers such fias methyleyclohexanone sand 'zdimethylcyclohexanone.

The x-invention also consists min processessfor the solvent:extractionof penicillin :c'arriedi =out substantially in 3.00OflGSJHCGWitIHiEDyiOf thesev- -er'al examples g'ive'n' below.

' T-he following examples illustratehow' the :inven'tionmay' heycarriedinto eiiect.

oEacamzilci *ortliopho'sphorio-acid,-:swhich brought' the. acidity ;55?

of *the penicillin solutioni tma PI-I'FOfA. I The! resulting acidified"solution was :then' extracted with "200 c. -'c.' methyl cyclohexanone."eA-f ter separation" an amount-of peniciilinequivalent. to 950,000 O.*U. was found in the: methyl-acyclohexanone' layer and an amountequivalent to 59,0000. U. in the aqueousi'layer. The amount ofsolidsextracted' by the solveirtzwasd-AO) we, 'thus the potency of thesolidz materialaobtained from-the solvent 1 wasflequivalente cacao OUz/img. 1-00 Example 2 V "'20 c. c. of a solution of penicillin:containing an amount equivalent to 18,800 0. U. were acidified at 0 C.with 0.90 c. c. of an aqueous solution containing orthophosphoric-acid,thus imparting to the penicillin solution a pH of 3.9.

5 This solution was then extracted at 0 C. with i0 that originallypresent was found in the organic layer, wh lle' an"amount equivalent to900 O. U. s "was found. in: thaaqueous layer.

General 5 Although in carrying the present invention mising the lossesdue-to. inactivation (ofthepeni- 'cillin. aOnr-theotheri hand thisequipment is::expensiveaandzits handling-and: maintenance require care.-With solvents in accordance --With 1the present invention itisipossi-ble to work undeir com ditionsof acidity where 'pH'is"greater-than4 wherex :penic-illin is: comparatively :stahleyeonsequently-.'inthe-extraction equipment centrifuges can -be-replaced-.by settlingtanks; which require .a: longer separation time but are very much *lessexpensive.

{"40 1 We claim:

1. In the process of recovering penicillin from aqueous liquorscontainingi theesame, the. steps Which-comprise bringing: such an:aqueous liquor to'-a pHsrof' a'bout 2 tot, admixing therewith at leastone methyl cyclohexanone;;separatingtthe resulting organic solvent'layerzirom.:the1aqueous layerrandirecovering penicillin fromthe former.

2. The process of claim1'1wherein the methyl *cyhlohexanone :is'mono'=methyl cyclohexanone.

'3'. Theprocess of claim 1--=where1n thez meth'yl cy'c'lohexanone isdim'ethyl' cyclohexanone.

DERRICK 'ROVI'LEY,

HEE/BERT STEINER.

'05 EMANUEL ZI-MKIN.

REFERENGES CITED [Thefollowing references are of record inthe file .of'thispatent:

.Lancet, II,.Aug. 16,1941, pp. 177-189.

.Abraham: lBritishJ. of Experimental Pathology, vol'23,'June 1942, No.3, ,pp. 103-115.

